My long-term goal is to become a leading independent investigator heading a truly interdisciplinary group that integrates cutting-edge bioinformatic approaches with innovative wet-bench methodologies to better understand RNA-mediated regulation, particularly in the context of bacterial pathogenesis. In the mentored phase of this award, I created and employed a high-throughput computational program called SIPHT to identify and annotate regulatory RNAs (regRNAs) and utilized data from cDNA deep sequencing experiments to determine a comprehensive profile of the V. cholerae small transcriptome. In the independent phase of the award, I plan to improve upon these computational and experimental approaches and to extend their application to a wider variety of bacterial species. Aim I is to develop an improved version of SIPHT that automatically updates its databases to enable regRNA predictions and annotations in newly sequenced strains and to incorporate newly identified regRNAs into predictive searches. Aim II Is to use Iterative SIPHT searches and machine learning techniques to improve the reliability of regRNA predictions and annotations. The studies proposed in Aim III have two main objectives. The first is to utilize massively parallel sequencing of the M. tuberculosis, V. cholerae, and P. aeruginosa transcriptomes to identify novel regRNAs and to elucidate RNA-mediated regulatory responses to specific environmental stresses and cues. The second is to develop bioinformatic algorithms to identify novel regRNAs from within the large datasets produced by deep sequencing techniques. Finally, Aim IV is to improve upon current predictive algorithms for trans-acting small regRNAs (sRNAs) by determining a consensus motif for the binding sites of the RNA chaperone Hfq and utilizing this motif in the search for previously unknown sRNAencoding genes. The proposed studies will likely lead to the discovery and functional annotation of numerous regRNAs. Moreover, the approaches described will be useful in the study of RNA-mediated regulation in a wide variety of bacterial species. Numerous recent studies have implicated a role for regRNAs in mediating the virulence of bacterial pathogens. Thus, identifying and characterizing regRNAs In a wider variety of species, in addition to providing insights Into myriad basic biological processes, will likely lead to a better understanding of microbial pathogenesis that may ultimately aid in the prevention and treatment of infectious diseases.